Project Summary/Abstract In melanoma, loss of function mutations in NF1 are the third most common oncogenic alteration after activating mutations in BRAF and NRAS. The RAS GTPase-activating protein NF1 provides negative feedback from the MAPK pathway to RAS by binding to SPRED1 at the plasma membrane where it is inactivates RAS-GTP. We recently discovered recurrent inactivating mutations of SPRED1 in melanoma, occurring in a mutually exclusive pattern with inactivating NF1 mutations, consistent with their functional overlap. Inactivation of either tumor suppressor often co-occurs with additional MAPK activating mutations such as activating mutations of KIT. We hypothesize that NF1 or SPRED1 inactivation may reduce the efficacy of therapeutic inhibition of KIT and similar gain of function alterations in the MAPK pathway. The objective of this research is to identify the spectrum of additional MAPK activating alterations that occur in the setting of NF1 or SPRED1 loss and to determine the role of NF1 or SPRED1 in melanoma progression and response to targeted therapies. Our central hypothesis is that NF1 or SPRED1 loss increases MAPK signaling but that full malignant transformation requires additional alterations that feed into the MAPK pathway. We will catalogue the spectrum of cooperators that form oncogenic pairs with NF1 or SPRED1 inactivation and validate their contributions to tumorigenicity both in vitro and in vivo. We hypothesize that identifying oncogenic pairs and developing therapeutic strategies that address both members of a pair will lead to improved outcomes for patients. We will provide proof of principle for this concept by building on our preliminary data that dual inhibition of KIT and MEK are synergistic for melanomas with an oncogenic pair consisting of KIT mutation and NF1 or SPRED1 loss. We will test combination therapies in genetically engineered human melanoma cell lines and mouse models. We will also investigate the direct physical interaction between KIT and SPRED1, which may lead to additional avenues for inhibiting melanomas with KIT or SPRED1 mutations. This work will provide a framework for similar studies for other cooperators of NF1 and SPRED1. This research is significant since it will lead to novel therapies for a significant number of melanoma patients for whom targeted therapies are not currently available. The proposed research is innovative because it tests a novel hypothesis why treatment of mutations such as KIT have limited efficacy in melanoma. Shedding light onto the complex signaling pathway perturbations in the considerable share of melanomas with NF1 or SPRED1 will have a considerable impact for patients with melanomas for which treatment options are currently limited.